摘要
为分离鉴定马尾松毛虫幼虫肠道细菌,从中挖掘产蛋白酶的肠道细菌资源。以马尾松毛虫(Dendrolimus punctatus)幼虫为材料,通过常规细菌培养方法体外获得部分肠道细菌,利用16S rDNA鉴定细菌种类,采用酪蛋白选择培养基筛选具有产蛋白酶能力的菌株,依据GB/T 23527—2009《蛋白酶制剂》的检测方法,测定蛋白酶活性。结果表明:从马尾松毛虫肠道分离出18个不同种类的非厌氧细菌,隶属于厚壁菌门(Firmicutes)的芽孢杆菌属(Bacillus),变形菌门(Proteobacteria)的亚硫酸杆菌属(Sulfurobacillus)、葡萄球菌属(Staphylococcus)、嗜冷杆菌属(Psychrobacter)、泛菌属(Pantoea)、假单胞菌属(Pseudomonas)、不动杆菌属(Acinetobacter)和柠檬酸杆菌属(Citrobacter)。经酪蛋白选择培养基筛选后共获得6株产蛋白酶细菌,均为芽孢杆菌。酶活性测定结果显示:这些产蛋白酶细菌均具有较高的产蛋白酶能力,尤其是菌株MW-39地衣芽孢杆菌(Bacillus licheniformis),酶活性最高可达255.36 U·mL^-1;其次为MW-50解淀粉芽孢杆菌(Bacillus amyloliquefaciens)和MW-4枯草芽孢杆菌(Bacillus subtilis),酶活性最高可分别达193.85 U·mL^-1和165.81 U·mL^-1。分析3个蛋白酶活性最高菌种的蛋白酶酶学特性,发现它们均具有一定的热稳定性和酸碱耐受性。单因素方差分析结果显示,在pH 5.0~9.0和30~50℃各菌株产生的蛋白酶活性相对较高,MW-39、MW-4和MW-50的蛋白酶最适pH分别为8.0、7.5、8.0,最适温度分别为45、40、40℃。本研究揭示了马尾松毛虫幼虫肠道多个细菌种类,丰富了产蛋白酶细菌资源,为进一步开发利用昆虫肠道产蛋白酶细菌资源提供了参考。
To isolate and identify intestinal bacteria from Dendrolimus punctatus larvae and to tap resources of protease-producing bacteria.D.punctatus larvae was used as experimental material in this paper,several intestinal bacteria were obtained by conventional bacterial culture method in vitro.16S rDNA was used to identify these bacteria,and protease-producing strains were screened by casein protein selection medium.Protease activity was determined according to GB/T 23527—2009“protease preparation”test method.The results showed that 18 different species of non-anaerobic bacteria were obtained,including Bacillus genera belonging to Firmicutes,and Sulfurobacillus,Staphylococcus,Psychrobacter,Pantoea,Pseudomonas,Acinetobacter and Citrobacter belonging to Proteobacteria.Six strains of the protease-producing bacteria all belonging to Bacillus genus were screened by casein selective medium.The results of enzyme activity showed that all of these protease-producing bacteria had high protease-producing ability,especially strain MW-39(Bacillus licheniformis),the highest enzyme activity reached to 255.36 U·mL^-1,followed by MW-50(Bacillus amyloliquefaciens)and MW-4(Bacillus subtilis),their highest enzyme activity reached to 193.85 U·mL^-1 and 165.81 U·mL^-1,respectively.Enzymatic characteristics of protease produced by three bacterial species with the highest enzymatic activity were further analyzed,and their proteases showed a certain thermal stability and acid-base tolerance.One-way ANOVA analysis showed that protease activity of investigated bacteria were high at pH 5.0-9.0 and 30-50℃,and the optimum pH of protease produced by MW-39,MW-4 and MW-50 were 8.0,7.5 and 8.0,respectively,and the optimum temperatures were 45℃,40℃and 40℃.This study revealed various species of intestinal bacteria in larvae of D.punctatus,enriched resources of protease-producing bacteria,and could provide reference for further development and utilization of protease-producing bacteria in insect intestine.
作者
江宇航
李宏伟
蔡赛波
林连兵
张棋麟
JIANG Yuhang;LI Hongwei;CAI Saibo;LIN Lianbing;ZHANG Qilin(Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, China;Engineering Research Center for Replacement Technology of Feed Antibiotics of Yunnan College, Kunming 650500, China)
出处
《浙江农业学报》
CSCD
北大核心
2020年第8期1446-1456,共11页
Acta Agriculturae Zhejiangensis
基金
国家自然科学基金(31960286)
云南省教育厅科学研究基金(2019J0050)。
关键词
马尾松毛虫
肠道细菌
芽孢杆菌
蛋白酶
酶学特性
Dendrolimus punctatus
intestinal bacteria
Bacillus
protease
enzymological characteristics
